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. 2006 Aug;26(15):5784-96.
doi: 10.1128/MCB.00232-06.

Priming-dependent phosphorylation and regulation of the tumor suppressor pVHL by glycogen synthase kinase 3

Alexander Hergovich  1 , Joanna Lisztwan, Claudio R Thoma, Christiane Wirbelauer, Robert E Barry, Wilhelm Krek
Affiliations

Priming-dependent phosphorylation and regulation of the tumor suppressor pVHL by glycogen synthase kinase 3

Alexander Hergovich et al. Mol Cell Biol. 2006 Aug.

Abstract

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is linked to the development of tumors of the eyes, kidneys, and central nervous system. VHL encodes two gene products, pVHL30 and pVHL19, of which one, pVHL30, associates functionally with microtubules (MTs) to regulate their stability. Here we report that pVHL30 is a novel substrate of glycogen synthase kinase 3 (GSK3) in vitro and in vivo. Phosphorylation of pVHL on serine 68 (S68) by GSK3 requires a priming phosphorylation event at serine 72 (S72) mediated in vitro by casein kinase I. Functional analysis of pVHL species carrying nonphosphorylatable or phosphomimicking mutations at S68 and/or S72 reveals a central role for these phosphorylation events in the regulation of pVHL's MT stabilization (but not binding) activity. Taken together, our results identify pVHL as a novel priming-dependent substrate of GSK3 and suggest a dual-kinase mechanism in the control of pVHL's MT stabilization function. Since GSK3 is a component of multiple signaling pathways that are altered in human cancer, our results further imply that normal operation of the GSK3-pVHL axis may be a critical aspect of pVHL's tumor suppressor mechanism through the regulation of MT dynamics.

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Figures

FIG. 1.
FIG. 1.
Endogenous pVHL regulates MT stability in a primary cell line. (A) Infected RPTEC cells were selected for 2 weeks before analysis of cell lysates by Western blotting with anti-pVHL(h)CT (top panel) and anti-Cdk2 (bottom panel) antibody. The positions of pVHL30 and pVHL19 species are highlighted by arrowheads. LMP, LTRmiR30-PIG; ns, nonsilencing; wt, wild type (untreated). (B) RPTECs expressing control (left panels) or miR30-based shRNA directed against pVHL (right panels) were processed for immunofluorescence using an anti-acetyl-tubulin antibody (red) before (top panels) or after (bottom panels) treatment with 10 μM nocodazole for 30 min. DNA is stained in blue. (C) Transfected cells were scored for the presence of stable MTs before and after treatment of cells with nocodazole. The experiment was done in triplicate; each bar represents a total of at least 400 cells analyzed. Error bars, standard deviations of the triplicates. pVHL-depleted cells displayed a significantly lower number of cells with intact MTs (white bar with asterisk).
FIG. 2.
FIG. 2.
Primed phosphorylation on serine 72 allows GSK3 phosphorylation of pVHL on serine 68 in vitro. (A) Alignment of amino acids 58 to 82 of pVHL30 with known GSK3 substrates. Serines 68 and 72 of pVHL30 are consistent with the GSK3 motif [(S/T)XXXSp]. Boldface indicates predicted phospho-residues. Underlining shows a putative GSK3 site, and italics indicate predicted priming sites. Serines 65, 68, 72, and 80 are indicated and exist in pVHL30 and pVHL19. (B) Purified GST-pVHL30 protein (Sf9 expressed) was incubated with kinase buffer alone (lane 1) or GSK3 (lanes 2 and 4). As a control, GSK3 was incubated in kinase buffer alone (lane 3). GST-pVHL30 was incubated with λ-phosphatase (λ-PPTase) before the phosphatase was washed out and the kinase assay was carried out (lane 5). wt, wild type. (C) Purified bacterially expressed GST-pVHL30 was incubated with CKI (lanes 1 and 3) or PKA (lane 2). GST-pVHL30(S68A) or -(S72A) was also incubated with CKI (lanes 4 and 5). (D) Bacterially expressed GST-pVHL30 was either incubated with GSK3 alone (lanes 1, 7, and 8) or sequentially incubated with first CKI and then GSK3 (lanes 2 to 6). The GSK3 inhibitor (inh.) 361535 was added at a concentration of 100 μM (lane 4). GST-pVHL30(S68A) or -(S72A) was not phosphorylated by GSK3 (lanes 5 and 6). GST-pVHL30(S72D) was a good (lane 7) and GST-pVHL30(S68A/S72D) a poor (lane 8) GSK3 substrate without prior CKI phosphorylation. (E) S68-phosphorylated, S72-phosphorylated, or nonphosphorylated peptides were spotted onto a membrane before immunoblotting using an anti-pVHL(S68-P) (top) or an anti-pVHL(S72-P) antibody (bottom). (F) Bacterially expressed GST-pVHL30 was either incubated with CKI (lane 2) or GSK3 (lane 3) or sequentially incubated with first CKI and then GSK3 (lanes 3 to 6). Proteins were analyzed by immunoblotting using an anti-pVHL(S68-P) (top), anti-pVHL(S72-P) (center), or anti-pVHLCT (bottom) antibody. (G) Phospho-S72 (black) or nonphosphorylated (gray) peptides were incubated with GSK3 for the indicated times before incorporated radioactivity was analyzed in counts per minutes. In panels B, C, and D, proteins were separated by SDS-PAGE and visualized either by autoradiography (upper panel) or Coomassie staining (lower panel).
FIG. 3.
FIG. 3.
Phosphorylation of pVHL by GSK3 in vivo. (A, B, and C) Lysates of COS-7 cells expressing the indicated cDNAs were processed for immunoprecipitation (IP) using an anti-HA antibody before analysis by Western blotting with anti-pVHL(S68-P) (top) or an anti-HA (center) antibody. Input lysates were immunoblotted using an anti-GSK3β antibody (bottom). (D) Cell lysates were processed and analyzed as described above, but input lysates were further immunoblotted with an anti-α-tubulin antibody. The positions of pVHL30 and pVHL19 species are highlighted by arrowheads. The anti-HA antibody light chain is indicated by an asterisk. (E) IMCD-3 cells were grown to confluence, then serum starved (no FCS) for 48 h and incubated in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of 20 mM LiCl for 4 h before processing for immunoprecipitation using an anti-pVHL(m)CT (lanes 2 and 3) or a control (lanes 1 and 4) antibody. Immunoprecipitates were analyzed by Western blotting with an anti-pVHL(S68-P) (top) and an anti-pVHL (Ig32) (center) antibody. Input lysates were immunoblotted using an anti-pVHL(m)CT antibody (bottom). The positions of mouse pVHL25 and pVHL21 species are highlighted by arrowheads. A nonspecific band is indicated by an asterisk.
FIG. 4.
FIG. 4.
GSK3β negatively affects pVHL's microtubule stabilization function without altering pVHL's microtubule association. (A) COS-7 cells transiently expressing HA-tagged pVHL30 and the indicated GSK3β constructs were treated with nocodazole before triple staining with anti-HA (blue), anti-GSK3β (green), and anti-α-tubulin (red) antibodies. White arrows indicate transfected cells. (B) Transfected cells were scored for intact MTs after treatment of cells with nocodazole. The percentage of transfected cells with intact MTs is plotted. Error bars, standard deviations for three independent experiments. Active GSK3β downregulated pVHL's MT stabilization function (red bars), in contrast to inactive GSK3β (green bars). (C) Whole-cell extracts (L) of COS-7 cells transfected with the indicated HA-tagged pVHL30 and GSK3β constructs were prepared and incubated with (lanes 4, 5, 9, and 10) or without (lanes 2, 3, 7, and 8) taxol-stabilized MTs. Supernatant (S) and pellet (P) fractions were processed for Western blotting with an anti-HA (left panels) and an anti-GSK3β (right panels) antibody.
FIG. 5.
FIG. 5.
The subcellular localization of pVHL30 is not affected by GSK3. (A) U2-OS cells were either left untreated or incubated with 20 mM LiCl or NaCl before processing for immunofluorescence with an anti-pVHL30 (green) and an anti-α-tubulin (red) antibody. DNA stainings are in blue. (B) U2-OS cells expressing empty vector, wild-type GSK3β [GSK3β(wt)], or kinase-dead GSK3β [GSK3β(kd)] were processed for immunofluorescence using an anti-pVHL30 (green), an anti-α-tubulin (red), and an anti-GSK3β (blue) antibody. Colocalization of pVHL30 with MTs appears yellow in the merged pictures.
FIG. 6.
FIG. 6.
Serines 68 and 72 of pVHL are required for the regulation of pVHL's microtubule function by GSK3β. (A) COS-7 cells coexpressing the indicated cDNAs were treated with nocodazole before triple staining with anti-HA (blue), anti-GSK3β (green), and anti-α-tubulin (red) antibodies. White arrows indicate transfected cells. (B) The percentage of cells coexpressing pVHL and GSK3 with intact MTs is plotted. Error bars, standard deviations for three independent experiments. (C) The percentage of cells expressing pVHL derivatives alone with intact MTs is plotted. Error bars, standard deviations for two independent experiments.
FIG. 7.
FIG. 7.
Residues 54 to 94 of pVHL are required for the regulation of pVHL's microtubule function by GSK3β. (A) Schematic representation of pVHL and mutant derivatives. The domain involved in MT binding (residues 95 to 123) is indicated. (B) COS-7 cells were first double transfected with the indicated HA-tagged deletion constructs/point mutants of pVHL30 and untagged wild-type GSK3β cDNAs and then scored for intact MTs after nocodazole treatment. The percentage of transfected cells with intact MTs is plotted. Error bars, standard deviations from at least two independent experiments. Dark gray bars indicate mutants unaffected by GSK3β.
FIG. 8.
FIG. 8.
pVHL's microtubule function is regulated by the priming-dependent kinase activity of GSK3. (A) COS-7 cells coexpressing the indicated cDNAs {wild-type GSK3β [GSK3β(wt)], kinase-dead GSK3β [GSK3β(K85R)], priming-dependent activity-deficient GSK3β [GSK3β(R96A)], and unprimed activity-deficient GSK3β [GSK3β(L128A)]} were treated with nocodazole before processing for triple staining with anti-HA, anti-GSK3β, and anti-α-tubulin antibodies. The percentage of transfected cells with intact MTs is plotted. Error bars, standard deviations for three independent experiments. (B) Cells were either left untreated (−) or preincubated with LiCl (+) before nocodazole was added. Selected mutants are shown in purple (S68D and S68D S72D), red (S72D), or green (S68A S72D). (C) In parallel, lysates of cells expressing the indicated HA-pVHL30 species were processed for immunoblotting using an anti-HA (α-HA) (top), anti-GSK3β (center), or anti-α-tubulin (bottom) antibody. (D) Lysates of cells expressing the indicated HA-pVHL30 species were processed for immunoprecipitation (IP) using an anti-HA antibody, followed by immunoblotting with an anti-pVHL(S68-P) (top panel) or anti-HA (second panel) antibody. Input lysates were immunoblotted using an anti-GSK3β antibody (third panel) or an anti-α-tubulin antibody (bottom panel). Cells were either left untreated (−) or preincubated with LiCl (+).
FIG. 9.
FIG. 9.
pVHL30(S68D/S72D) forms an E3 ligase complex but does not bind to HIF2α. (A) 786-O cell pools stably expressing empty vector, wild-type pVHL30, or pVHL30 harboring different amino acid substitutions at S68 and S72 were processed for immunoblotting using an anti-HIF2α (top panel), anti-Glut-1 (second panel), anti-pVHL30 (third panel), anti-α-tubulin (fourth panel), or anti-Cdk2 (bottom panel) antibody. (B) Whole-cell extracts of 786-O cell pools were subjected to immunoprecipitation with an anti-pVHL (Ig32) antibody, and immunoprecipitates were analyzed by immunoblotting using an anti-Cul2 (top) or anti-pVHL30 (center) antibody. In parallel, input lysates were processed for Western blotting with an anti-Cul2 antibody (bottom). (C) Sf9-expressed and purified GST-pVHL30 was incubated with kinase buffer alone (lane 1) or recombinant GSK3 (lane 2). After kinase reaction, glutathione-Sepharose-bound proteins were washed with lysis buffer before incubation with equal amounts of in vitro-translated, HA-tagged HIF2α. Finally, unbound HIF2α was washed out, and glutathione-bound proteins were analyzed by immunoblotting using an anti-HA (top) or anti-GST (bottom) antibody.
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